Antibodies are extremely valuable, both as therapeutic agents and as general reagents in a variety of molecular biological processes. Methods of producing polyclonal and monoclonal antibodies are available, as are many antibodies. A number of basic texts describe standard antibody production processes, including, e.g., Borrebaeck (ed) (1995) Antibody Engineering, 2nd Edition Freeman and Company, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford, England (McCafferty), and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, N.J. (Paul); Paul (ed.), (1993) Fundamental Immunology, Raven Press, N.Y.; Coligan (1991) Current Protocols in Immunology Wiley/Greene, N.Y.; Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature 256: 495-497.
Naturally occurring antibodies, or immunoglobulins (Igs), comprise a basic four polypeptide chain structure comprising two identical heavy (H) chains and two identical light (L) chains which are stabilized and cross-linked by intrachain and interchain disulphide bonds. Different antibody classes comprise variants of this four-chain structure. Each heavy chain comprises a variable domain at N-terminal followed by several constant domains. Each light chain has a variable domain at its N-terminal and one constant domain at its C-terminal. Because the largest amount of sequence variation is concentrated in the N-terminal domains of the light and heavy chains, each of these domains is termed a variable (V) domain (or “V region”). The constant domains make up the constant region, which comprises the remainder of the molecule and exhibits relatively little sequence variation. Heavy chains are comprised of five major types, depending on the antibody class, and consist of about 450-600 amino acid residues. Light chains are of two major types and have about 230 amino acid residues. Both heavy and light chains are folded into domains, comprising globular polypeptide regions.
In the antibody, the variable domain of the light chain is aligned with the variable domain of the heavy chain; the constant domain of the light chain is aligned with the first constant domain of heavy chain. The variable domains of each pair of light and heavy chains form the antigen binding site for binding the antibody to an epitope of the antigen. The constant domains in the light and heavy chains are not directly involved in antigen binding. Each heavy or light chain variable domain comprises four relatively conserved framework (FR) regions (or framework segments) which are separated and connected by three hypervariable or complementarity determining regions (CDRs), which are believed to contact the target antigen of the antibody and to be principally responsible for binding of the antibody to the antigen.
The framework regions and CDRs have been precisely defined. See, e.g., Kabat, E. A. et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, US Dept. Health and Human Services, National Institutes of Health, USA (5th ed. 1991); and Wu et al., J. Exo. Med. 132:211-250 (1970), each of which is incorporated herein by reference in its entirety for all purposes. For additional discussion of the structure of variable domains, see Poljak, R. J. et al., PNAS USA, 70, 3305-3310, 1973; Segal, D. M. et al., PNAS USA, 71, 4298-4302, 1974; and Marquart, M. et al., J. Mol. Biol., 141, 369-391, 1980, each of which is incorporated herein by reference in its entirety for all purposes. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The combined heavy and light chain framework regions of an antibody serve to position and align the CDRs for proper binding to the antigen.
The amino acids of the CDRs of the variable domains were initially defined by Kabat, based on sequence variability, to consist of amino acid residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the human heavy chain variable domain (VH) and amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the human light chain variable domain (VL), using Kabat's numbering system for amino acid residues of an antibody. See Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, US Dept. Health and Human Services, NIH, USA (5th ed. 1991). Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) presented another definition of the CDRs based on residues that included in the three-dimensional structural loops of the variable domain regions, which were found to be important in antigen binding activity. Chothia et al. defined the CDRs as consisting of amino acid residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the human heavy chain variable domain (VH), and amino acid residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the human light chain variable domain (VL). Combining the CDR definitions of Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (H1), 50-65 (H2), and 95-102 (H3) in human VH and amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in human VL, based on Kabat's numbering system.
V genes encode the approximately N-terminal 95 amino acids of the V domains. The number of V genes at each locus varies between loci and species, but may include up to about several hundred V genes.
Antibody heavy chain V domains include V genes, D (diversity) genes, and J (joining) genes. The large diversity in antibody variable domains results from, in part, recombination between V, D, and J gene segments. To produce a gene encoding a heavy chain variable domain, any one of the heavy chain variable domain genes is recombined with any one of a small number of D and J genes to produce a VDJ gene. The recombination process of a light chain variable domain is similar, except that a V gene is recombined directly with a J gene, since light chain variable domains have no D gene segments.
Over the last decade, a variety of recombinant techniques for antibody preparation which do not rely on injection of an antigen into an animal have been developed. For example, it is possible to generate and select libraries of recombinant antibodies in phage or similar vectors. See, e.g., Winter et al. (1994) “Making Antibodies by Phage Display Technology” Annu. Rev. Immunol. 12:433-55 and the references cited therein for a review. See also, Griffiths and Duncan (1998) “Strategies for selection of antibodies by phage display” Curr Opin Biotechnol 9: 102-8; Hoogenboom et al. (1998) “Antibody phage display technology and its applications” Immunotechnology 4: 1-20; Gram et al. (1992) “in vitro selection and affinity maturation of antibodies from a naïve combinatorial immunoglobulin library” PNAS 89:3576-3580; Huse et al. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546. Kits for cloning and expression of recombinant antibody phage systems are known and available, e.g., the “recombinant phage antibody system, mouse ScFv module,” from Amersham-Pharmacia Biotechnology (Uppsala, Sweden). Bacteriophage antibody libraries have also been produced for making high affinity human antibodies by chain shuffling (Marks et al. (1992) “By-Passing Immunization: Building High Affinity Human Antibodies by Chain Shuffling” Biotechniques 10:779-782.
In general, the libraries include repertoires of V genes (e.g., harvested from populations of lymphocytes or assembled in vitro) which are cloned for display of associated heavy and light chain variable domains on the surface of filamentous bacteriophage. Phage are selected by binding to an antigen. Soluble antibodies are expressed from phage infected bacteria and the antibody can be improved, e.g., via mutagenesis. For example, Stemmer et al. (1993) “Selection of an Active Single Chain Fv Antibody From a Protein Linker Library Prepared by Enzymatic Inverse PCR” Biotechniques 14(2):256-65 describes production of large libraries of site directed single chain Fv antibody mutants. Other references also propose library mutagenesis strategies, such as computer assisted oligo directed scanning mutagenesis. See, e.g., Balint and Larrick (1993) “Antibody Engineering by Parsimonious Mutagenesis” Gene 137:109-118.
More recently, forced evolution methods have been adapted to recombinant antibody construction and improvement methods to produce optimized antibodies. For example, Crameri et al. (1996) “Construction and evolution of antibody-phage libraries by DNA shuffling” Nature Medicine 2:100-103 describe, e.g., the construction and evolution of antibody-phage libraries by a variety of DNA recombination procedures, e.g., DNA shuffling. Crameri and Stemmer (1995) “Combinatorial multiple cassette mutagenesis creates all the permutations of mutant and wildtype cassettes” BioTechniques 18:194-195 describe, e.g., in vitro recombination of antibody DNA fragments (scFv fragments) by combinatorial multiple cassette mutagenesis.
A variety of patents by the inventors and their co-workers provide additional details on diversification procedures, e.g., shuffling, applicable to the directed evolution of antibodies, including U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “METHODS FOR IN VITRO RECOMBINATION;” U.S. Pat. No. 5,811,238 to Stemmer et al. (Sep. 22, 1998) “METHODS FOR GENERATING POLYNUCLEOTIDES HAVING DESIRED CHARACTERISTICS BY ITERATIVE SELECTION AND RECOMBINATION;” U.S. Pat. No. 5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA MUTAGENESIS BY RANDOM FRAGMENTATION AND REASSEMBLY;” U.S. Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10, 1998) “END-COMPLEMENTARY POLYMERASE REACTION,” and U.S. Pat. No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “METHODS AND COMPOSITIONS FOR CELLULAR AND METABOLIC ENGINEERING.”
For example, the '793 patent details improvement of antibodies involving diversification, e.g., by DNA shuffling, of a library of mutant CDRs. The '721 patent details, e.g., peptide display methods and antibody display and screening methods. In general, a variety of antibody diversification are found throughout the noted patents.
Although methods of producing antibodies by making, screening and evolving antibodies and antibody libraries are established, it would be desirable to have additional techniques for antibody generation and refinement. Furthermore, a general technology platform for addressing these issues would be desirable. The present invention provides these and other features which will be apparent upon a complete review of the following.